Method for the light-controlled synthesis of biochips

ABSTRACT

The present invention relates to a method for the photolithographic synthesis of biochips in which photolabile protective groups of the 2-(2-nitrophenyl)ethyl type are used, whereby the irradiation step that is common in the photolithographic chip synthesis is carried out in the presence of a base, preferably a secondary or uerriary base.

This application is a National Stage of International ApplicationPCT/DE99/04051, filed Dec. 17, 1999; which claims the priority of DE 19858 440.7, filed Dec. 17, 1998.

FIELD OF THE INVENTION

The present invention relates to a method for the light-controlled(photolithgaphic) synthesis of biochips.

BACKGROUND OF THE INVENTION

The field concerning the synthesis of oligonucleotides and DNA or RNAchips has been booming for several years, and efficient methods arerequired in this connection.

Synthetic DNA fragments (oligonucleotides) are nowadays prepared almostexclusively in accordance with to the phosphite amide method. Thispreparation is made with the aid of what is called DNA synthesizers.After inputting the target sequence, these machines automaticallysynthesize the desired DNA oligomer. For this purpose, the DNAsynthesizer has a computer-controlled programming determining in whichorder certain reagents are pumped onto the synthesis column where theactual synthesis of the DNA oligomer takes place in the solid phase onglass beads of e.g. controlled pore glass (CPG). The reagents includeinter alia the 4 (protected) phosphite amides for the 4 bases (adenine,cytidine, guanosine, thymine), the catalyst (usually tetrazole), anoxidizing agent (usually iodine), capping reagent A & B (usually aceticanhydride, N-methylimidazole), and detritylation reagent (usuallytrichloroacetic acid). A reaction cycle consists of the followingsequence:

(1) detritylation cleavage of the temporary 5′-O-dimethoxytritylprotecting groups by trichloroacetic acid; (2) condensation thephosphite amide corresponding to the target sequence is condensed ontothe released 5′-hydroxyl function; this is done with the aid of theacidic catalyst (tetrazole); (3) oxidation the unstable phosphitetriester linkage (P-III) is converted by oxidation with iodine into astable phosphorus triester (P-V); (4) capping the 5′-hydroxyl functionswhich did not react in the preceding step shall be trapped by anacetylation step (acetic anhydride + N-methylimidazole); this shallprevent uncontrolled growth of the DNA strand.

Steps (1) to (4) are then repeated until the target sequence has beenobtained.

The initiator nucleoside, which carries preferably a5′-O-dimethoxytrityl protecting group, is usually found on the supportalready, so that the detritylation step is started. As soon as thetarget sequence has been obtained, the phosphate protecting groups andthe protecting groups of the exocyclic amino functions of theheterobases have to be cleaved. This is usually done by means ofammonia. The cleavage of the oligomer from the support also takes placesimultaneously with ammonia, so that the fully synthesized oligomer isthen in the ammonia cleavage solution. Having evaporated the ammoniasolution, the target oligomer is obtained.

The step decisive for the quality of the synthesis is the condensation,since all of the other steps (detritylation, oxidation, capping) proceedquantitatively. The condensation is carried out in the absolute absenceof humidity. Condensation yields of up to 99% can be achieved.

The synthesis of nucleic acid chips on support surfaces is carried outaccording to an analogous method, so that, in principle, the belowdescribed chip synthesis methods are also suitable for the preparationof oligonucleotides if they are removed from the support surface.

Some time ago, another method was developed for the nucleic acid-chipsynthesis: the light-controlled chip synthesis. The phosphite amides (A,C, G, T) used in the light-controlled synthesis have the same chemicalstructure as in the “normal” DNA synthesis on a commercial DNAsynthesizer, the only difference being that the 5′-acid-labiledimethoxytrityl protecting group is replaced by a photolabile protectinggroup which may be at position 5′ or 3′. This leads to the followingreaction course on the DNA chip synthesizer:

(1) Irradiation cleavage of the temporary photolabile protecting groupby irradiation using light of corresponding wavelength; (2) condensationthe phosphite amide corresponding to the target sequence is condensedonto the released hydroxyl function; this is done with the aid of theacidic catalyst (tetrazole); (3) oxidation the unstable phosphitetriester linkage (P-III) is converted by oxidation with iodine into astable phosphorus triester (P-V); (4) capping the hydroxyl functionswhich did not react in the preceding step shall be trapped by anacetylation step (acetic anhydride + N-methylimidazole); this shallprevent uncontrolled growth of the DNA strand.

Steps (1) to (4) are then repeated until the target sequence has beenobtained.

The initiator nucleoside is usually not found on the support, so thatthe condensation step is started with here. As soon as the targetsequence has been obtained, the phosphate protecting groups and theprotecting groups of the exocyclic amino functions have to be split offas well.

This is done by means of ammonia but under milder conditions (2 h) sothat the DNA strands synthesized on the chip are not split off thesupport. This becomes possible by using more labile protecting groupsthan employed for the common synthesis of CPG materials as protectinggroups of the exocyclic amino functions. The DNA chip is then simplyremoved from the ammoniacal solution, washed with water and canimmediately be used for hybridization experiments.

The quality of the DNA chip synthesis is determined in thelight-controlled method not only by condensation alone but, above all,by the efficiency of the cleavage of the photolabile protecting groupswhich only in rare cases is as effective as the cleavage of thedimethoxytrityl protecting group by means of acid. Since yields of up to95-99% can usually be achieved in the condensation step, the quality ofthe DNA chip is determined more or less by the efficiency of thephotoprotecting group cleavage.

WO 96/18634 and WO 97/44345 are special photolabile protecting groups ofthe 2-(2-nitrophenyl)ethyl type which shall be suitable for thepreparation of oligonucleotides on a DNA chip. However, the inventorshave already found out that it is very difficult to split off thephotolabile protecting groups shown in these applications by commonmethods and that the preparation of DNA or RNA chips is not veryefficient. Moreover, it turned out that due to their inferior qualitychips prepared in such a way cannot be detected by means offluorescence, which is current standard to detect DNA chips. Probelabeling by means of radiography would be required for the detection ofthese chips. This is, however, not usable for commercial exploitation.

The object of the present invention consists in providing a method bymeans of which high-quality biochips, in particular DNA or RNA chips,can be produced.

This object is achieved by the subject matters defined in the claims.

SUMMARY OF THE INVENTION

The present invention is achieved by a method for the light-controlledbiochip synthesis in which photolabile protecting groups of the2-(2-nitrophenyl)ethyl type are used. The irradiation step common in thelight-controlled chip synthesis is carried out in the presence of abase.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the U.S. Patent and TrademarkOffice upon request and payment of the necessary fee.

FIG. 1 shows the reaction scheme for the DNA-chip synthesis for buildingup an 8-mer (CAGGTCGC) on a glass support surface,

FIG. 2 shows the NPPOC photochemistry; conventional method (without baseaddition or not enough base) results in no DNA chip synthesis.

FIG. 3 shows the NPPOC photochemistry; irradiation with the addition ofbases results in effective DNA chip synthesis.

DETAILED DESCRIPTION OF THE INVENTION

The method according to the invention for the light-controlled biochipsynthesis offers the advantage that the efficient cleavage ofphotolabile protecting groups of the 2-(2-nitrophenyl)ethyl type can beachieved. The photolabile protecting groups have preferably thefollowing formula:

R¹=H, NO₂, CN, OCH₃, halogen, alkyl or alkoxyalkyl having 1 to 4 C atoms

R²=H, OCH₃, alkyl residue having 1 to 4 C atoms or optionallysubstituted aryl residue

R³=H, F, Cl, Br, NO₂, alkyl residue having 1 to 4 C atoms or optionallyan aryl residue or an aliphatic acyl residue having 2 to 5 C atoms,

R⁴=H, halogen OCH₃, alkyl residue having 1 to 4 C atoms or optionallysubstituted aryl residue

R⁵=H, NO₂, CN, OCH₃, halogen, alkyl or alkoxy alkyl having 1 to 4 Catoms or optionally substituted aryl residue

X=SO₂ (sulfonyl), OCO (oxycarbonyl)

It is particularly preferred to use the protecting groups NPPOC or CNPOCwhich have the following formula, NPPOC being most preferred:

The above protecting groups may be used generally for protectinghydroxyl or amino functions on nucleic acid derivatives, peptides,proteins, antibodies or other biomolecules, preferably for protectingthe hydroxyl functions in nucleic acid derivatives, in particular forprotecting the 5′-OH position or the 3′-OH position in nucleic acidderivatives. As mentioned already in connection with the above priorart, they shall be used in particular for the synthesis of high-qualityDNA or RNA chips, which has not yet succeeded since the step ofphotodeprotection by irradiation does not proceed efficiently enough.High-quality DNA chips are meant to be those obtained when the yield ineach step is as high as possible during the light-controlled synthesis.

According to the invention a biochip is understood to mean biomolecules,such as DNA, RNA, nucleic acid analogs (e.g. PNA, LNA), proteins,peptides, antibodies, synthesized on a support, the three former groupsbeing preferred.

According to the invention any support or matrix, common in this field,can be used for the biochip production. These are in particular glass,sheets or membranes made of polypropylene, nylon, cellulose, cellulosederivatives (e.g. cellulose acetate, mixed cellulose phosphate group. Ina preferred embodiment the support surfaces have a derivatizationaccording to German patent application 198 53 242.3.

The steps condensation, oxidation and capping are carried out as usualin the method for light-controlled biochip synthesis according to theinvention (Fodor et al., Science 1991, 251, page 767 et seq.). Accordingto the invention the first step of synthesis, namely the irradiation, ishowever carried out by adding bases, preferably strong bases, inparticular non-nucleophilic bases, which in cooperation with the lightused for the irradiation leads to a surprisingly effective cleavage ofthe protecting groups. The invention provides for the fact that byadmixing bases during the irradiation a higher yield can be achieved onthe solid support material. In this connection, it must be stressed thatan efficient cleavage of the protecting groups on the solid supportmaterial cannot be achieved by either the influence of light withoutbase addition or base addition alone. Correspondingly, the methodaccording to the invention makes use of a well defined system consistingof light action in combination with base addition for a successfullight-controlled parallel synthesis of DNA arrays on the solid supportmaterial.

For example, the irradiation in the presence of 0.05 Mdiisopropylethylamine in acetonitrile represents such an exemplarysuccessful combination of light action and base addition. The NPPOCphotoprotecting group cannot be cleaved efficiently by irradiation invarious solvents without base addition (e.g. in acetonitrile alone) norcan it be abstracted by treatment with 0.05 M diisopropylethylamine inacetonitrile without the action of light. This means that only thecombination of a base together with the action of light effects anefficient cleavage of the protecting groups.

In a preferred embodiment bases, such as DBU(1,8-diazabicyclo[5.4.0]undec-7-ene), diisopropylethylamine,N-methylmorpholine, N-methylimidazole, piperidine are suitable asaddition during the irradiation. The secondary or tertiary bases knownto the person skilled in the art, e.g. DBN(1,5-diazabicyclo[4.3.0]none-5-ene), triethylamine, DABCO, 2,6-lutidine,collidine, morpholine, diisopropylamine, triethylamine, are also usable.

The irradiation may take place under common conditions. The wavelengthof irradiation depends on the protecting group used. The person skilledin the art is familiar with the suitable wavelengths. A 5-minuteirradiation with a 100 W Hg lamp at a wavelength between 320 and 380 nm,preferably at 365 nm, is suitable for splitting off the NPPOC or CNPOCprotecting group.

The amount of base present during the irradiation varies between 0.01 Mand 1.0 M and depends, of course, on the base strength. It provedefficient to use 0.03 to 1 M (preferably 0.05 to 0.5 M) DBU inacetonitrile, 0.03 to 0.8 M (preferably 0.05 M) diisopropylethylamine inacetonitrile or 0.03 to 1 M (preferably 0.5 M) piperidine inacetonitrile.

In other preferred embodiments, modifications occur in the other stepsof photolithographic nucleic acid chip synthesis. It turned out to bepreferable to use pyridine hydrochloride (preferably 0.1 to 1.0 M, morepreferably 0.5 M, in actonitrile) in place of tetrazole as a catalystfor the condensation. Moreover, it was found that the formerly commonaddition of THF can be dispensed with for the oxidizing agent. Areduction of the common iodine amount also proved advantageous for theoxidizing step. Furthermore, the common THE was replaced by acetonitrilein the capping reagent. As can be proved, the capping procedure mayfully be omitted without a negative effect on the quality of the chipsresulting. Thus, the synthesis can be accelerated considerably.

The invention is described in more detail by means of the below example:

EXAMPLES Example 1 Light-controlled (Photolithographic) DNA-chipSynthesis Using NPPOC as a Protecting Group

The DNA chip synthesis was carried out in analogy to the known method byFodor et al. (Science 1991, 251, p. 767 et seq.) on a glass surface asthe support. The reaction course is shown in FIG. 1 by way of diagram.NPPOC-protected phosphite amides (company of Nigu-Chemie, Waldkraiburg)were used as nucleosides.

A DNA chip synthesizer program is composed of individual subroutineswhich correspondingly represent the individual synthesis steps for theoligonucleotide synthesis according to the phosphite amide chemistry andare processed one after the other. The cycle is as follows:

(1), (3), (5), (9), (12) = M-wash subroutine which is interposed betweenall of the other ones for the purpose of washing the reaction flowchamber and removing residues from the preceding step (2) = M-couplinkage of the new phosphite amide building block on the photo-released5′-OH function of the preceding building block at this position (3) =M-ox labile phosphite triester function is oxidized by iodine to givethe phosphate triester (6) = M-dry actuates the opening of the shutterand thus the irradiation of the reaction flow chamber using light. Anexternal electronic circuit which is operated by the DNA synthesizer viaa potential-free signal actuates the opening. This circuit serves forsetting how long the shutter remains open and thus how long irradiationtakes place. The irradiation procedure is described in more detail below(7), (10) = M-irrad. The irradiation solution is pumped into thereaction flow chamber. In some cases, the irradiation solution containsa base (DBU, piperidine, . . .) (8), (11) = M-waitdr. wait loop of 110sec. (may be adjusted as desired), both subroutines together yield adesired irradiation period (here: 5 minutes) (1) M-wash (2) M-coup (3)M-wash (4) M-ox (5) M-wash (6) M-dry (7) M-irrad. (8) M-waitdr. (9)M-wash (10) M-irrad. (11) M-waitdr. (12) M-wash

The irradiation (5 minutes of irradiation with a 100 W Hg lamp, at 365nm) was carried out as follows:

opening of the shutter (thus starting of irradiation in the reactionchamber),

flushing of the reaction flow chamber with irradiation solution,

the irradiation solution is pumped out of the chamber after 150 seconds,

short washing of the chamber using acetonitrile,

another flushing of the reaction flow chamber with irradiationsolution—after a total of 300 seconds the shutterdcloses (theirradiation is terminated),

the irradiation solution s pumped out of the chamber,

thorough washing of the chamber using acetonitrile.

The subsequent steps of condensation, oxidation and capping remainedunchanged as compared to the reaction course of the prior art (Weiler etal, Analytical Biochemistry, 243, pp. 218-227 (1996)) or showed themodifications described on page 8.

The irradiation solutions contained 0.05 M DBU, 0.5 M DBU, 0.05 Mdiisopropylethylamine or 0.05 M piperidine, each in acetonitrile.

Chips using NPPOC-protected phosphite amides but without using base orwith using not enough base in the irradiation step were produced as acontrol. The irradiation solutions contained in these cases 0.005 M DBUin acetonitrile, pure acetonitrile or 10% H₂O/MeOH. In one case, theirradiation was carried out in a dry state.

When comparing FIGS. 2 and 3, which represent a fluorescence analysis ofthe produced chips, it has to be noted that in the irradiation aneffective DNA chip synthesis is only possible with base additive, i.e. apattern corresponding to mask II can only be detected there. No chipsynthesis is obtained when there is no or not enough base addition,which is expressed by the totally blackened images. It is thus provedthat the addition of the base contributes actively to the abstraction ofthe photo-protecting group of the type of formula (I).

What is claimed is:
 1. A method for synthesizing a light-controlled biochip comprising the step of carrying out an irradiation step common for a photolithographic chip in the presence of a base, wherein photolabile protecting groups of the 2-(2-nitrophenyl)ethyl type are used.
 2. The method according to claim 1, wherein the base is a secondary or tertiary base.
 3. The method according to claim 1 or 2, wherein the base is selected from the group consisting of 1, 8-diazabicycloundec-7-ene, 1, 5-diazabicycionone-5-ene, diisopropylethylamine, pyridine, piperidine, triethylamine, diisopropylamine, N-methylmorpholine, 2,6-lutidine, collidine, N-methylimidazole, DABCO, and N,N-dimethylaminopyridine.
 4. The method according to claim 3, wherein the base is used in a concentration of 0.01 to 1 M.
 5. The method according to claim 1 or 2, wherein the photolabile protecting group has the formula (I):

R¹=H, NO₂, CN, OCH₃, halogen, alkyl or alkoxyalkyl having 1 to 4 C atoms, R²=H, OCH₃, alkyl residue having 1 to 4 C atoms or optionally substituted aryl residue, R³=H, F, Cl, Br, NO₂, alkyl residue having 1 to 4 C atoms or optionally an aryl residue or an aliphatic acyl residue having 2 to 5 C atoms, R⁴=H, halogen, OCH₃ alkyl residue having 1 to 4 C atoms or optionally substituted aryl residue, R⁵=H, NO₂, CN OCH₃, halogen, alkyl or alkoxyalkyl having 1 to 4 C atoms or optionally substituted aryl residue, and X=SO₂ (sulfonyl), OCO (oxycarbonyl).
 6. The method according to claim 5, wherein the photolabile protecting group is NPPOC or CNPOC.
 7. The method according to claim 1 or 2, wherein the biochip is a nucleic acid chip or nucleic acid analog chip.
 8. The method according to claim 7, wherein the nucleic acid chip is a DNA or RNA chip.
 9. The method according to claim 7, wherein the nucleic acid analog chip is a PNA or LNA chip.
 10. The method according to claim 1, further comprising carrying out a condensation step common in a light-controlled chip synthesis with pyridine hydrochloride as a catalyst. 